Dopamine provides reward for adaptive behaviors such as food and sexual activity, but drugs of abuse release dopamine directly, bypassing the normal regulatory mechanisms and producing long-term changes that result in tolerance, physical dependence and drug craving. The long-term objective of this proposal is to understand how dopamine release is regulated, and how changes in release influence behavior and contribute to drug abuse. The strategy is to use vesicular monoamine transporter 2 (VMAT2) as a tool to characterize the exocytotic release of monoamines, and to study it as a locus for regulation. Unlike most other classical neurotransmitters, monoamines including dopamine undergo release from cell body and dendrites as well as the axpn terminal. Importantly, somatodendritic dopamine release is required to induce behavioral sensitization, a model for drug-seeking. However, the mechanism of somatodendritic dopamine release remains unclear, with some evidence favoring vesicular release and other reverse flux through the dopamine transporter. In Specific Aim 1, we will assess the potential for exocytotic release by studying the trafficking of VMAT2, the carrier required for loading vesicles with monoamine. Preliminary data suggests that somatodendritic vesicles containing VMAT2 undergo regulated exocytosis, and we will now use both fixed cells and live cell imaging to characterize the exocytosis and endocytosis of VMAT2 in live neurons, and to identify the signals responsible for sorting VMAT2 to this pathway. We will then use this information to manipulate the localization of VMAT2 in vivo, test unambiguously the mechanism of somatodendritic dopamine release, and determine the role of somatodendritic release in the plasticity of monoamine systems in general. In Specific Aim 2, we will use VMAT2 to address basic questions about the relationship between vesicle filling and the recycling of membrane in the synaptic vesicle cycle. Exploring the biochemical basis for a genetic interaction identified in C. elegans, we have found that the v-SNARE synaptobrevin required for regulated exocytosis inhibits the activity of VMAT2, and this effect does not reflect changes in transporter expression or trafficking. Rather, the v-SNARE appears to inhibit VMAT2 directly. We will now characterize the mechanism for this inhibition, and use this information to determine the role of this interaction in dopamine release. The results will provide physiologically relevant information about a novel mechanism regulating vesicular neurotransmitter transport that may contribute to the long-term changes in dopamine release that accompany behavioral sensitization.